This invention relates to embedding media for the preparation of thin sections of embedded biological materials. The thin sections are of the type which may be employed for light or electron microscopic study of the embedded biological materials.
U.S. Pat. No. 4,424,329 describes producing embedding media by admixing several methacrylates, a free radical polymerisation initiator, one or more co-initiators and--optionally--a plasticizer. The use of a complex initiator system instead of a simple compound is, however, a disadvantage. Apart from this, the described initiator system is not optimal for polymerisation at low temperature.
Described in J. Electron Microscopy 28, (1979) pp. 53-55, is that tree cell walls can be best observed by embedding tree cells in a medium consisting of methyl and n-butyl methacrylate and polymerizing the embedding medium. However, polymerisation is carried out only at elevated temperature with 2,2'-azobisisobutyronitrile as an initiator. Furthermore, the two-component embedding medium lacks variability.
Finally, a method of producing an embedding medium which comprises admixing several methacrylates and a free radical polymerisation initiator is described in DE-A No. 27 48 938. However, this embedding medium is likewise polymerised at elevated temperature and additionally admixing the monomer mixture with a further component of polymer powder is required Furthermore, the preparation of biological specimens with available embedding media by the thin sectioning technique, although usually adequate for general cytological work, has severe limitations for high resolution electron microscopy as in the molecular region.
In order to improve the amount of useful information retrievable, a number of deleterious effects must be minimised, including the effects of molecular denaturation, supramolecular disordering, damage by sectioning, and other factors related to observation, such as staining, and beam damage due to irradiation in the electron microscope.
Most attempts to improve embedding techniques in the past have involved the consideration that a water-soluble resin renders dehydration essentially unnecessary and that use of such resins will not lead to solvent-induced denaturation. What does not appear to have been properly observed or considered is that the liquid resin itself can be a solvent which leads to solvent denaturation. Furthermore, the fact that a liquid resin may be water-soluble does not ensure that the initial polar environment of cell structures would be maintained.
Procedures for the preparation of thin sections of embedded biological materials are in general characterized by the inability to independently alter several parameters, including particularly:
the effect on biological materials such as proteins and lipoproteins when water is replaced by organic fluids employed in the embedding procedure,
the effect of polarity of the organic solvent and embedding medium,
effects of temperature,
effects of water content remaining after dehydration,
effects of the type and duration of fixation,
effect of the nature of heavy metal staining.
It is an object of the present invention to provide new embedding media enabling parameters such as above to be varied independently. This enables determination of the influence of each parameter on the different structural aspects in biological materials such as proteinaceous complexes and protein-rich lipoprotein membranes. The parameters are extremely interdependent or intercorrelated, as for example illustrated by the fact that aminoacid residues at the surface of proteins as well as polar heads of lipids are firmly associated with a layer of water, the hydration shell, and the fact that the binding of this water on proteins is different for different molecular surfaces and can "melt away" at different temperatures. Thus, the higher this "melting temperature", the "firmer" is the water bound. This phenomenon is involved in the formation of the hydrophobic bond, which becomes established only upon raising of temperature. Such bonds are entropy driven, i.e. the disorder associated with "melting" of the hydration water increases the entropy more than the association (ordering) proteins does decrease it. Many believe that the hydration shell is an important factor in establishing the tertiary structure of a protein. Pertubing or removing it, would lead to the conformational changes associated with denaturation. Therefore, the fate of the hydration shell during embedding must be considered. Similarly, with highly polar organic solvents or embedding media, in which water is soluble, the possibility of competitive effects between these liquids and the biological material for the remaining water of hydration must be considered. Particularly in a polar resin, a sort of composition might occur which removes the hydration shell, if water has a higher affinity to the resin. Since no experimental data are as yet available, this question has to be solved empirically for the embedding. Non-polar solvents or embedding media on the contrary have no affinity for water and it can be envisaged that the hydration shell is not removed, provided obviously that this was maintained during dehydration. This returns the consideration to those related to polar solvents.
Two embedding medium compositions, which take into account the considerations discussed above are described in U.S. Pat. No. 4,424,329. However, the viscosity of these compositions is a limiting factor for application at lower temperatures. To avoid these disadvantages, three new compositions two polar and one non-polar, have been developed, which enable embedding at a substantially lower temperature range.